The current emphasis is to understand first the role of lysosomal membrane protein 1, LAMP1, in cytotoxicity of human NK cells. To address this issue, RNAi was used to disrupt LAMP1 expression and assess its effects on lytic granule exocytosis and NK cell cytotoxic potential. We generated YTS and NK92 NK cell lines with a stable LAMP1 knockdown (LAMP1 KD) (80% decrease at protein level). With siRNA-mediated silencing of LAMP1 in ex vivo isolated IL-2 cultured human NK cells, we achieved a 40% to 50% decrease of LAMP1 protein. The lower LAMP1 knockdown level chieved in the transiently siRNA-transduced ex vivo NK cells was at least partially due to the stability of LAMP1; LAMP1 has a half-life of at least 30 hours, and the time point at which NK cells were analyzed (72 hours) was likely too short for stronger or full silencing of LAMP1. To investigate the role of LAMP1 in NK-cell function, we evaluated the effect of LAMP1 silencing on NK-cell cytotoxicity. We found that compared with untransduced YTS cells, or cells transduced with non-targeting control RNAi, LAMP1 RNAi cells had a 70% to 76% decrease in cytotoxicity against tumor cell lines (721.221 or K562). We obtained similar results using ex vivo isolated NK cells, where a transient knockdown of LAMP1 reduced cytotoxicity by 40% to 60%. We also used Ab-dependent cell-mediated cytotoxicity to evaluate the killing ability of LAMP1 RNAi NK cells. Although CD16-mediated recognition of anti-Human Epidermal Growth Factor Receptor 2 (HER2) Ab, bound to the cell surface of SK-OV3 cells, resulted in pronounced lysis of target cells by control-transduced NK cells, cytotoxicity by LAMP1 RNAi NK cells was decreased by 35% to 40%. Our results demonstrate for the first time that LAMP1 plays a critical role in NK cell cytolytic activity. Furthermore, we found that, in contrast to control RNAi-transduced cells, LAMP1 RNAi cells failed to deliver granzyme B to 721.221 target cells. Similarly, siRNA-mediated KD of LAMP1 in ex vivo NK cells also blocked the transfer of granzyme B from NK to tumor cells. Importantly, LAMP1 RNAi cells (both YTS and ex vivo NK cells) had normal level of granzyme B mRNA and protein, and the activity of granzyme B from LAMP1 RNAi cells was undistinguishable from the activity in control cells. LAMP1 RNAi cells had normal conjugation, and were able to externalize CD107b on the cell surface, excluding the possibility that the block of granzyme B delivery and, subsequently, cytotoxicity resulted from improper cell-cell adherence or inhibition of fusion of lytic granules with the plasma membrane. Our results suggested that the defect of granzyme B delivery could be due to problems with granule transport of lytic granules to the cell to cell contact site. Laser scanning confocal microscopy experiments showed that although LAMP1 RNAi cells were able to translocate lytic granules toward the immunological synapse, the granules were more dispersed around the MTOC and did not cluster efficiently at the immunological synapse, suggesting a possible defect in granule transport in LAMP1 RNAi cells. Indeed, we discovered that compared to control RNAi cells, granules in LAMP1 RNAi cells traveled shorter distances, had smaller displacement and moved slower, indicating that LAMP1 is important for the proper granule movement along cytoskeletal tracks. Using cell fractionation and analytical ultracentrifugation, we found that, when compared to control RNAi cells, lytic granules isolated from LAMP1 KD cells had less p150glued, an important component of the microtubule motor protein complex-dynein/dynactin. The decreased recruitment of motor proteins to lytic granules provides an explanation for the defects in the movement of the granules caused by the disruption of LAMP1 expression. More importantly, we also found that compared to control RNAi cells, silencing of LAMP1 resulted in 30-40% decrease of perforin fluorescence, despite normal level of perforin mRNA. Analysis of intracellular perforin level by flow cytometry confirmed the specific decrease of perforin in ex vivo NK and YTS cells with LAMP1 KD. Imaging and lytic granule isolation studies led us to discovery that LAMP1 RNAi cells had less perforin associated with lytic granules. Consequently, we found that the decreased level of perforin in lytic granules was due to the defect in perforin trafficking, and retention of perforin in the trans-Golgi (TGN)-derived transport vesicles. The adaptor protein complex AP-1, important for sorting proteins leaving the TGN, is known to directly interact with LAMP1. Therefore, we analyzed the effect of silencing the AP-1 complex subunit, adaptin gamma, on perforin location in YTS cells. Adaptin gamma silencing resulted in increased association of perforin with the transport vesicles. Importantly, adaptin gamma knock-down produced effects similar to LAMP1 silencing, indicating that these two binding partners are important for proper traf&#64257;cking of perforin to lytic granules and NK-cell cytotoxicity. Our data suggest that the direct interaction between LAMP1 and AP-1 could mediate transport of LAMP1-positive vesicles, containing perforin, to lytic granules. The lack of LAMP1 could prevent binding of AP-1 to perforin-positive vesicles and result in retention of perforin in the transport vesicles instead of its delivery to lytic granules. Similarly, the lack of AP-1 complex would prevent proper perforin transfer from the TGN to lytic granules because there would be no adaptor to interact with LAMP1 and enable the transport. We have shown that disruption of LAMP1 expression has a pleiotropic effect on NK cells, causing impairment of traf&#64257;cking of a critical lytic granule protein, perforin, and slower movement of lytic granules. This results in the inability of NK cells to deliver granzyme B to target cells and inhibition of NK-cell cytotoxicity. Thus, LAMP1 is not only a marker of NK-cell degranulation, but also a crucial component of NK-cell cytotoxicity.